Observation of the Blood-Brain Barrier Opening by Ultrasound with Microbubbles on Mice Using Intravital Imaging with Two-photon Microscopy

Low-intensity focused ultrasound (FUS) has been shown to temporarily increase the permeability of the blood-brain barrier (BBB) in the presence of circulating microbubbles (MB), facilitating the delivery of therapeutic agents to the brain. It is important to understand the short-term or long-term effect of FUS/MB on cerebrovascular health and the dynamic diffusion process of therapeutic agents in the brain interstitium in vivo. In this study, an ultrasound treatment platform was established to observe the FUS/MB-mediated BBB opening utilizing two-photon fluorescence imaging with both temporal and spatial resolution in real time. First, cranial window chamber models were established. The BBB disruption was visualized by the application of Evans blue (EB) dye and two types of leakage pattern were observed. One was fast leakage that the EB extravasation occurred drastically and quickly in less than one minute during the sonication. The other was slow leakage that the extravasation of EB generally happened several minutes after sonication and lasted a relative long time slowly and continuously.